pGEX-2TK

Bacterial vector for expressing GST fusions with a thrombin site and a cAMP kinase site.

Sequence Author: GE Healthcare

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HomePlasmidspGEX Vectors (GE Healthcare)pGEX-2TK
BtgI (4869) Bsu36I (4761) PluTI (4311) SfoI (4309) NarI * (4308) KasI (4307) HpaI (4174) EcoRV (4118) BssHII (4079) ApaI - BanII (3879) PspOMI (3875) BstEII (3849) MluI (3668) BstAPI (3351) PflMI (3250) AlwNI (2643) BfuAI - BspMI (62) lac operator EcoNI (268) MscI (465) BstBI (655) SwaI (685) pGEX 5' Sequencing Primer (869 .. 891) kinase site BamHI (951) AvaI - BsoBI - TspMI - XmaI (956) SmaI (958) MCS EcoRI (961) PfoI (1036) pGEX 3' Sequencing Primer (1019 .. 1041) PflFI - Tth111I (1139) BsaAI (1146) ZraI (1243) AatII (1245) PstI (1922) BsaI (2098) AhdI (2164) pGEX-2TK 4969 bp
BtgI  (4869)
1 site		 C C R Y G G G G Y R C C
Sticky ends from different BtgI sites may not be compatible.
Bsu36I  (4761)
1 site		 C C T N A G G G G A N T C C
Sticky ends from different Bsu36I sites may not be compatible.
PluTI  (4311)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (4309)
1 site		 G G C G C C C C G C G G
NarI  (4308)
1 site		 G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (4307)
1 site		 G G C G C C C C G C G G
HpaI  (4174)
1 site		 G T T A A C C A A T T G
EcoRV  (4118)
1 site		 G A T A T C C T A T A G
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
BssHII  (4079)
1 site		 G C G C G C C G C G C G
BssHII is typically used at 50°C, but is 75% active at 37°C.
ApaI  (3879)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
BanII  (3879)
1 site		 G R G C Y C C Y C G R G
Sticky ends from different BanII sites may not be compatible.
PspOMI  (3875)
1 site		 G G G C C C C C C G G G
BstEII  (3849)
1 site		 G G T N A C C C C A N T G G
Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
MluI  (3668)
1 site		 A C G C G T T G C G C A
BstAPI  (3351)
1 site		 G C A N N N N N T G C C G T N N N N N A C G
Sticky ends from different BstAPI sites may not be compatible.
PflMI  (3250)
1 site		 C C A N N N N N T G G G G T N N N N N A C C
Sticky ends from different PflMI sites may not be compatible.
AlwNI  (2643)
1 site		 C A G N N N C T G G T C N N N G A C
Sticky ends from different AlwNI sites may not be compatible.
BfuAI  (62)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (62)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
EcoNI  (268)
1 site		 C C T N N N N N A G G G G A N N N N N T C C
The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MscI  (465)
1 site		 T G G C C A A C C G G T
BstBI  (655)
1 site		 T T C G A A A A G C T T
SwaI  (685)
1 site		 A T T T A A A T T A A A T T T A
SwaI is typically used at 25°C, but is 50% active at 37°C.
BamHI  (951)
1 site		 G G A T C C C C T A G G
After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
AvaI  (956)
1 site		 C Y C G R G G R G C Y C
Sticky ends from different AvaI sites may not be compatible.
BsoBI  (956)
1 site		 C Y C G R G G R G C Y C
Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
TspMI  (956)
1 site		 C C C G G G G G G C C C
XmaI  (956)
1 site		 C C C G G G G G G C C C
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (958)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
EcoRI  (961)
1 site		 G A A T T C C T T A A G
PfoI  (1036)
1 site		 T C C N G G A A G G N C C T
Sticky ends from different PfoI sites may not be compatible.
PflFI  (1139)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (1139)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BsaAI  (1146)
1 site		 Y A C G T R R T G C A Y
ZraI  (1243)
1 site		 G A C G T C C T G C A G
AatII  (1245)
1 site		 G A C G T C C T G C A G
PstI  (1922)
1 site		 C T G C A G G A C G T C
BsaI  (2098)
1 site		 G G T C T C N C C A G A G N ( N ) 4
Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (2164)
1 site		 G A C N N N N N G T C C T G N N N N N C A G
The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
pGEX 5' Sequencing Primer
23-mer  /  65% GC
1 binding site
869 .. 891  =  23 annealed bases
Tm  =  66°C
pGEX 3' Sequencing Primer
23-mer  /  65% GC
1 binding site
1019 .. 1041  =  23 annealed bases
Tm  =  66°C
lacI
3318 .. 4400  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
3318 .. 4400  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
1377 .. 2237  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   1377 .. 1445  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1377 .. 2237  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   1446 .. 2237  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1377 .. 2237  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GST
258 .. 911  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
GST
258 .. 911  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from Schistosoma japonicum
thrombin site
918 .. 935  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
918 .. 935  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
kinase site
936 .. 950  =  15 bp
5 amino acids  =  587.7 Da
Product: cAMP-dependent protein kinase recognition site
kinase site
936 .. 950  =  15 bp
5 amino acids  =  587.7 Da
Product: cAMP-dependent protein kinase recognition site
ori
2408 .. 2996  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2408 .. 2996  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
1272 .. 1376  =  105 bp
AmpR promoter
1272 .. 1376  =  105 bp
lacIq promoter
3240 .. 3317  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
3240 .. 3317  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
tac promoter
183 .. 211  =  29 bp
3 segments
   Segment 1:  -35  
   183 .. 188  =  6 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
183 .. 211  =  29 bp
3 segments
   Segment 2:  
   189 .. 204  =  16 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
183 .. 211  =  29 bp
3 segments
   Segment 3:  -10  
   205 .. 211  =  7 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
183 .. 211  =  29 bp
3 segments
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
lac operator
219 .. 235  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
219 .. 235  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
MCS
951 .. 966  =  16 bp
multiple cloning site
MCS
951 .. 966  =  16 bp
multiple cloning site
stop codons
971 .. 981  =  11 bp
stop codons in all three reading frames
stop codons
971 .. 981  =  11 bp
stop codons in all three reading frames
ORF:  3251 .. 3568  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  4157 .. 4420  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4523 .. 81  =  528 bp
ORF:  175 amino acids  =  19.7 kDa
ORF:  258 .. 977  =  720 bp
ORF:  239 amino acids  =  27.7 kDa
ORF:  1377 .. 2237  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3441 .. 4400  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4908 .. 178  =  240 bp
ORF:  79 amino acids  =  9.1 kDa
ORF:  1841 .. 2107  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  4183 .. 4434  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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